Microsoft Word - 7- Dr Kalantar RTL 89 10 1_2_.doc Iran J Arthropod-Borne Dis, 2010, 4(2): 50–55 B Davari et al.: Frequency of Resistance and … 50 Original Articles Frequency of Resistance and Susceptible Bacteria Isolated from Houseflies B Davari1, *E Kalantar2, A Zahirnia3, SH Moosa-Kazemi4 1Department of Parasitology, School of Medicine, Medical University of Kurdistan, Sananadaj, Iran 2Liver and Digestive Research Center, Tohid Hospital, Medical University of Kurdistan, Sananadaj, Iran 3Department of Parasitology, School of Medicine, Medical University of Hamedan, Hamedan, Iran 4Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Iran (Received 3 Mar 2010; accepted 4 Jul 2010) Abstract Background: In this study, we determine the vector competence of Musca domestica with reference to the transmis- sion of susceptible and resistance bacterial strains in hospitals and slaughter house in Sanandaj City, west Iran. Methods: Totally 908 houseflies were collected to isolate bacteria from their external body based on standard proce- dures.Antibiotic susceptibility testing was performed by Kirby-Bauer disc diffusion method on Mueller Hinton agar based on recommendations of CLSI (formerly the National Committee for Clinical Laboratory Standards). Results: From collected houseflies, 366 bacteria species were isolated. The most common isolated bacterium at hos- pitals was Klebsiella pneumoniae 43.3% (n= 90) followed by Pseudomonas aeruginosa 37% (n= 77), while that of slaughterhouse was Proteus mirabilis. 29.1% (n= 46) followed by Citrobacter freundii 28.4% (n= 45). Among all the isolates from hospitals, cephalexin, chloramphenicol, ampicillin, and tetracycline, resistance rates were above 32.5% and gentamicin expressed the highest susceptibility among all the isolates from hospitals. It is worth to note that K. pneumoniae showed 61% and 44.5% resistance to cephalexin and chloramphenicol respectively. Similarly, all iso- lates from slaughterhouse were more than 28% and 30% resistant to cephalexin and chloramphenicol respectively. Surprisingly, among all the isolates, Citrobacter freundii were highly resistant to gentamicin. Conclusion: Houseflies collected from hospitals and slaughterhouse may be involved in the spread of drug resistant bacteria and may increase the potential of human exposure to drug resistant bacteria. Keywords: House fly, bacterium, antibacterial resistance, hospitals, slaughterhouse Introduction The common house fly, Musca domes- tica is a medically-important insect world- wide (Fotedar 2001, Graczyk et al. 2001, Kabkaew et al. 2007). Houseflies have been implicated as vectors or transporters of vari- ous human pathogens, including Vibrio chol- erae, Enterobacteriaceae pathogens, Staphy- lococcus aureus, and Pseudomonas spp. (Ol- sen 1998, Fotedar 2001, Rajendran and Pan- dian 2003). Transmission takes place when the fly makes contact with people or their food. As many as 500000 microorganisms may swarm over its body and legs (Thiru- malai Vasan et al. 2008). “Flies can spread diseases because they feed freely on human food and dirty matter alike. The fly picks up disease-causing or- ganisms while crawling and feeding. The diseases that flies can transmit include en- teric infections, eye infections, poliomyelitis and certain skin infections.” Thus, houseflies are widely recognized as potential reservoirs and vectors of food borne pathogens (Pan- dian and Asumtha 2001, Khobdel et al. 2008). *Corresponding author: Dr Enayat Kalantar, Email: kalantar_enayat@yahoo.com Iran J Arthropod-Borne Dis, 2010, 4(2): 50–55 B Davari et al.: Frequency of Resistance and … 51 It is worth to note that a few studies also in- dicate that houseflies have been suspected to be reservoirs and vectors for pathogens (Za- rin et al. 2007, Barin et al. 2010). There have been no studies on the car- riage of antibiotic-resistant, pathogenic bac- teria by M. domestica in hospitals and slaughterhouse in Sanandaj. An increasing frequency of antibiotic resistance has been reported from all over of the world. In this regard, an attempt was made to determine the frequency of resistance and susceptible bacteria isolated from houseflies in Sanan- daj, west of Iran. Materials and Methods Houseflies were captured by a sterile nylon net from the wards and corridors of the Tohid Hospital, Beassat Hospital, and the slaughterhouse of the city. The collected flies were transferred immediately to the Entomology Laboratory, and identified to species level by morphological characters such as thorax, wings and antenna. After identification, 1 ml of sterile physiological saline solution was added to each vial, which was shaken vigorously for 1 min with the fly remaining inside. The fly was then removed from the saline, and was checked for bacteria dislodged from the ex- ternal surfaces of the fly. Bacterial Counts Serial dilutions of a subsample of each bacterial suspension were prepared in sterile saline. Each dilution was then inoculated onto two plates of plate-count agar and incu- bated overnight at 37 °C. Colony forming units (CFU/ ml) were then counted so that the total numbers of bacteria recovered from the external surface of each fly could be es- timated. Briefly, houseflies individually were shaken thoroughly in sterile saline solution (2 ml) for 2 min. The suspension was then serially diluted and inoculated on MacCon- key agar, and Blood agar. Plates were incu- bated for 24 h at 37 °C. The resulting isolates were characterized morphologically and further identifications were carried out following the methods of Koneman et al. 1992. Antibiotic susceptibility test was per- formed by Kirby-Bauer disc diffusion me- thod on Mueller Hinton agar based on rec- ommendations of CLSI (formerly the Na- tional Committee for Clinical Laboratory Standards) (NCCLS, 2003). The following antibiotics were used in this study: erythro- mycin, streptomycin, ampicillin, tetracycline, kanamycin ,chloramphenicol, co-trimoxazole, gentamicin, ciprofloxacin, nitrofurantoin, cef- triaxone, and cephalexin which were pur- chased from Patan Teb Company. Results From the 908 houseflies collected from the hospitals and slaughterhouse at Sanandaj, 366 (40.3%) bacterial species were isolated (Table 1). The most common bacterium isolated from M. domestica at hospitals was Kleb- siella pneumoniae 43.3% (n= 90) followed by Pseudomonas aeruginosa 37% (n= 77), while that of slaughterhouse was Proteus mirabilis 29.1 (n= 46) followed by Citro- bacter freundii 28.4% (n= 45) (Table 2). Among all the isolates from hospitals, cephalexin, chloramphenicol, ampicillin, and tetracycline, resistance rates were above 32.5% and gentamicin expressed the highest susceptibility among all the isolates from the hospitals. It is worth to note that K. pneumo- niae showed 61% and 44.5% resistance to cephalexin and chloramphenicol, respectively (Table 3). Similarly, all the isolates from slaughter house were more than 28%, 30% resistance to cephalexin and chloramphenicol respec- tively (Table 4). Surprisingly, among all iso- lates, Citrobacter freundii were highly resis- tant to cephalexin. Iran J Arthropod-Borne Dis, 2010, 4(2): 50–55 B Davari et al.: Frequency of Resistance and … 52 Table 1. Bacterial carrying rates for Musca domestica collected from hospitals and slaughter house at Sanandaj No. of bacterial species isolated from each fly Number isolates for habitats Hospitals Slaughter House Total Male Female Total Male Female Total Total positive flies 61 147 208 56 102 158 Total negative flies 78 132 210 127 205 332 Total flies examined 139 279 418 183 307 490 908 Table 2. Details of bacteria isolated from House Fly collected from hospitals and Slaughter house at Sanandaj Number isolates for habitats Bacteria Hospitals Slaughter house Number Percent Number Percent K. pneumoniae 90 43.3 23 14.5 P. aeruginosa 77 37.0 0.0 0.0 Citrobacter freundii 12 05.7 45 28.4 E. coli 19 09.1 26 16.4 Bacillus cereus 10 04.8 18 11.4 Proteus mirabilis 0.0 0.0 46 29.1 Total 208 100 158 100 Table 3. Antibiotic susceptibility pattern (%) of identified bacteria in hospitals at Sanadaj, Iran Bacteria K. spp. (90) P. aeruginosa (77) E. coli (19) B. cereus (10) Proteus mirabilisa (12) Antibiotic R I S R I S R I S R I S R I S Ery 46.6 31.1 11.1 39.0 36.3 24.6 26.0 42.1 31.5 20.0 60.0 20 16.0 41.6 41.6 Strep 38.8 34.4 26.6 32.5 41.5 25.9 31.0 26.3 42.1 30.0 50.0 20.0 33.0 66.6 0.0 Amp 43.3 38.8 17.7 35.0 45.4 19.4 31.0 26.3 42.1 30.0 40.0 30.0 25.0 33.3 41.6 Tetra 43.3 41.1 15.5 29.0 53.2 16.8 21.0 42.1 36.8 20.0 50.0 30.0 16.0 50.0 33.3 Kana 27.8 23.3 48.8 26.0 41.5 32.4 15.0 57.8 26.3 20.0 60.0 20.0 16.0 41.6 41.6 Chlo 44.5 46.6 08.8 32.5 23.3 44.1 31.0 42.1 26.3 20.0 70.0 10.0 16.0 58.3 25.0 Co-tri 35.5 31.1 33.3 31.0 32.4 36.3 26.0 63.1 10.5 10.0 50.0 40.0 08.3 33.3 58.3 Gen 11.0 16.6 72.2 09.0 46.7 44.1 15.0 42.1 42.1 20.0 50.0 30.0 16.0 50.0 33.3 Cipro 15.5 22.2 62.2 14.0 49.3 36.3 15.0 63.1 21.0 10.0 60.0 30.0 08.3 58.3 33.3 Nitrof 17.7 15.5 66.6 16.8 23.3 59.7 21.0 47.3 31.5 20.0 50.0 30.0 16.0 50.0 33.3 Ceftri 24.4 26.6 48.8 22.0 54.5 23.3 26.0 42.1 31.5 30.0 60.0 10.0 25.0 41.6 33.3 Cepha 61.0 31.1 07.7 44.0 28.5 27.2 42.0 47.3 10.5 40.0 50.0 10.0 33.0 50.0 16.6 Erythromycin, Streptomycin, Ampicillin, Tetracycline, Kanamycin ,Chloramphenicol, Co-trimoxazole, Gentamicin, Ciprofloxacin, Nitrofurantoin, Ceftriaxone , Cephalexin (S) = Sensitive, (I) = Intermediate, (R) = Resistant Iran J Arthropod-Borne Dis, 2010, 4(2): 50–55 B Davari et al.: Frequency of Resistance and … 53 Table 4. Antibiotic susceptibility pattern (%) of identified bacteria at slaughter house in Sanandaj, Iran Bacteria Klebsiella spp. (23) Proteus mirabilis (46) E. coli (26) Citrobacter freundii (45) Bacillus cereus (18) Antibiotic R I S R I S R I S R I S R I S Ery 21.7 43.4 34.7 15.0 43.4 41.3 19.0 61.5 19.2 15.4 17.7 66.0 11.1 33.3 55.0 Strep 26.0 30.4 43.4 15.0 52.1 32.6 23.0 69.2 07.6 24.4 22.2 53.0 11.1 22.2 66.0 Amp 21.7 26.0 43.4 17.0 60.8 21.7 27.0 46.1 23.0 20.3 17.7 62.0 22.2 27.7 50.0 Tetra 13.0 34.7 52.1 13.0 43.4 43.4 19.0 69.2 11.5 22.2 08.8 68.0 16.6 22.2 61.0 Kana 17.0 26.0 56.5 15.0 41.3 43.4 15.0 65.3 19.2 24.4 24.4 51.0 05.5 22.2 72.0 Chlo 30.0 34.7 34.7 14.0 60.8 26.0 23.0 57.6 19.2 15.5 15.5 68.0 11.1 33.3 55.0 Co-tri 30.0 21.7 47.8 06.5 52.1 41.3 27.0 53.8 15.3 06.6 11.1 82.0 16.6 38.8 44.0 Gen 08.6 56.5 34.7 13.0 43.4 43.4 15.0 46.1 38.4 08.8 13.3 77.0 0.0 22.2 77.0 Cipro 13.0 39.1 47.8 06.5 54.3 39.1 11.5 65.3 23.0 04.4 11.1 84.0 0.0 38.8 61.0 Nitrof 13.0 52.1 34.7 15.0 43.4 41.3 19.0 53.8 26.9 20.0 26.6 53.0 0.0 27.7 72.0 Ceftri 21.7 34.7 43.4 19.0 39.1 41.3 23.0 57.6 19.2 17.7 22.2 60.0 0.0 44.4 55.0 Cepha 47.0 30.4 21.7 28.0 45.6 26.0 30.0 65.3 03.8 66.0 20.0 13.3 05.5 33.3 61.0 Erythromycin, Streptomycin, Ampicillin, Tetracycline, Kanamycin, Chloramphenicol, Co-trimoxazole, Gentamicin, Ciprofloxacin, Nitrofurantoin, Ceftriaxone , Cephalexin Discussion “The biology and ecology of M. domes- tica make it an ideal mechanical vector of human and animal pathogens. Cattle barns, poultry houses, slaughter houses, and hospi- tals are sites where house flies can reproduce” (Peter et al. 2007). Many scientists indicated that the exter- nal organs of M. domestica (legs, wings and mouthparts) constituted a large source of bacteria they isolated (Graczyk 1999, Mutsuo et al. 1999). The results of this study indi- cated that M. domestica could play a great role as a mechanical carrier of bacteria. In this study, most of the bacteria isolated were medically important, including K. pnenu- monae, P. aeruginosa, Proteus mirabilis. These findings agree with the results of Vazirianzadeh et al. (2008) in Ahvaz, which showed presence Escherichia coli, P. aerugi- nosa, and K. pneumonia on the housefly collected from slaughterhouse and zoo. Our results are in accordance with other reports which highlight the importance of houseflies in carrying various pathogenic bacteria particularly K. pneumoniae being the most important at USA and Iran (Thad- deus et al. 2001, Khalil et al. 1994). The house flies caught in hospitals carried patho- genic bacteria more often than those caught at slaughter house which is in contrast with Sulaiman et al. (2000) study. One of the most important problems facing global public health today is antim- icrobial resistance. The problem is most hor- rible in developing countries, where the bacterial infections causing human disease are also those in which emerging antibiotic resistance is most evident (Shears 2000, Kalantar et al. 2008). The resistance patterns of the K. pneu- moniae isolated in the present study are shown in Table 3 and 4. The K. pneumoniae isolated from the hospitals houseflies were more resistant to cephalexin as compared to that of isolated from the slaughterhouse. Fotedar et al. (1992) and Sramova et al. (1992) reported similar multiple-resistance to antibiotics, in Klebsiella spp. from house- flies in hospitals environments. Similarly, the resistance patterns of the P. aeroginosa from hospitals environment houseflies were frequently multiple-resistant, Iran J Arthropod-Borne Dis, 2010, 4(2): 50–55 B Davari et al.: Frequency of Resistance and … 54 with more than 32% of the isolates each be- ing resistant to erythromycin, streptomycin, ampicillin, and cephalexin. Therefore, house- flies trapped in hospitals may also participate more in the dispersion of antibiotic resis- tance into the environment. Multiple resis- tances to antibiotics are common among P. aeruginosa isolated from different clinical sources in Iran (Kalantar et al. 2009). The present study indicates that housefly M. domestica poses a possible health risk to communities proved that the isolated strains of bacteria were resistant to various antibi- otics. It is well-established fact that the re- sistance to various antimicrobials may be due to presence of some virulence gene, in- volvement of secretion machinery of multi drug efflux proteins, through mutations in bacterial genome or by gaining additional genes through horizontal gene transfer or by physiology dependent resistance (Mitchell et al. 2004, Rangrez et al. 2006). In conclusion, we report that houseflies collected in hospitals and slaughterhouse may be involved in the spread of drug resis- tant bacteria and may increase the potential for human exposure to drug resistant bacte- ria. It is recommended that suitable steps must be taken to control the flies and moni- tor the sensitivity pattern of the pathogens transmitted by the houseflies. Acknowledgements This work received financial support from Medical University of Kurdistan (Grant number 680). The authors declare that they have no conflicts of interest. 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